Plastids and mitochondria are double membrane-bound organelles found in eukaryotic cells. Chloroplasts, plastids containing the green pigment chlorophyll, are the most complex of the plant membranous organelles. Both chloroplasts and mitochondria specialize in the synthesis of ATP, using energy derived from electron transport from photosynthetic phosphorylation in chloroplasts and from oxidative phosphorylation in mitochondria.
To perform their role in the cell, plastids must continuously import all types of molecules, including proteins. The biogenesis and development of plastids require the coordinated assembly of plastidic- and nuclear-encoded proteins which are incorporated into membranes or other parts of the plastid. The process by which nuclear-encoded plastid proteins are targeted from the site of synthesis to the site of finction is mediated by a complex series of events involving a multitude of proteinaceous signals and factors located in the cytosol and the plastidic compartment. Few of these factors are known and the molecular infrastructure underlying this important and complex event is far from being understood.
Chloroplast envelope proteins play a major role in modulating the vectorial flow of molecules across the membrane, including large proteinaceous entities. The import of proteins into the plastid is a complex process requiring the close collaboration of both the outer envelope and the inner envelope membranes. Evidence for the possible existence of two distinct protein import complexes, one in each envelope membrane, is beginning to emerge from a number of recent investigations (Waegemann, K. and Soll, J. (1991) Plant J. 1: 149-158; Soll, J. and Waegemarn, K. (1992) Plant J. 2:253-256; Schnell, D. and Blobel, G. (1992) J. Cell. Biol. 120:103-115; Alefson, H., Waegemann, K. and Soll, J. (1994) J. Plant Physiol. 144:339-345; Schnell, D., et al. (1994) Science 266:1007-1012; Kessler, F., et al. (1994) Science 266:1035-1039; Wu, C., Seibert, F. S. and Ko, K. (1994) J. Biol. Chem. 269:32264-32271).
An important step in the characterization of the protein translocating complexes is the identification of the components involved. The identification of outer and inner plastid envelope polypeptides has been accomplished using a variety of strategies (Ma, Y., et al. (1996) J. Cell Biol. 134:315-327; Comwall, K. L. and Keegstra, K. (1987) Plant Pysiol. 85:780-785; Kaderbhai, M. A., et al. (1988) FEBS Lett. 232:313-316; Pain, D., et al. (1988) Nature 331:232-237; Schnell, D., et al. (1990) J. Cell Biol. 111:1825-1838; Hinz, G. and Flugge, U.-I. (1988) Eur. J. Biochem. 175:649-659; Soll, J. and Waegemanm, K. (1992) Plant J. 2:253-256; Waegemann, K., et al. (1990) FEBS Lett. 261:89-92; Perry, S. E. and Keegstra, K. (1994) Plant Cell 6:93-105; Alefson, H., et al. (1994) J. Plant Pysiol. 144:339-345; Schnell, D. J., et al. (1994) Science 266:1007-1012; Kessler, F., et al. (1994) Science 266:1035-1039; Wu, C., et al. (1994) J. Biol. Chem. 269:32264-32271; Hirsch, S., et al. (1994) Science 266:1989-1992; Seedorf, M., et al. (1995) Plant J. 7:401-411; Seedorf, M. and Soll, J. (1995) FEBS Lett. 367:19-22; Gray, J. C. and Row, P. E. (1995) Trends Cell Biol. 5:243-247). To date, these studies collectively indicate that envelope proteins with molecular masses of 21, 30, 34, 36, 44, 45, 51, 66, 70, 75 86, 97 and 100 kDa may be possible constituents of the plastid protein import apparatus; however, it is not obvious from the existing data whether some of the predicted similar sized components are identical to each other. Further, it is not known if any of the components have an active role in protein transport.
A mechanism for controlling the transport of substances into plastids could be used for modification of plastid pathways and products which occur in particular tissue types, such as the starch and fatty acid biosynthesis pathways in roots and seeds. Major drawbacks to plastid modification of this caliber, however, are the limited knowledge of genes encoding plastid transport proteins and the lack of characterization of such proteins.
Further, plastid transport mechanisms could be usefully incorporated into other organisms, especially prokaryotes. The heterologous production of protein pharmaceuticals in Escheriehia coli is a cornerstone of the biotechnology industry. The technology provides an attractive and viable means for the production of proteins in quantities and qualities that are otherwise expensive and difficult to obtain from natural sources.
The gene sequence and encoded protein of one plastid membrane component has been identified. Ko, K., et al. (1995) J. Biol. Chem. 270:28601-28608; GenBank(trademark)/EMBL Data Bank, accession no. X79091. However, no role in transport was determined for this protein.
To date, no one has reported eukaryotic transport gene function or the functioning of a transport gene from a eukaryotic organelle in prokaryotic cells. An additional transport gene in both prokaryotic and eukaryotic cells would be useful to increase translocation and expression of cellular products. Increased incorporation of proteins into membranes to elevate membrane function would also be desirable.
This invention relates to a method for enhancing the transport of substances, particularly proteins, across a cellular membrane (xe2x80x9ctranslocationxe2x80x9d) by means of isolated or recombinant nucleic acids encoding a plastid transport protein (Bce44B) or its functional equivalent. Nucleic acids which hybridize to the Bce44B gene are also encompassed by this invention when such hybridizing sequences encode the functional equivalent of the Bce44B protein. The present invention also relates to a method for enhancing the incorporation of substances, particularly proteins, into cellular membranes.
The cellular membranes can be those of prokaryotic or eukaryotic cells. They can include membranes of organelles, either single- or double-membrane bound organelles, as well as plasma membranes.
One object of this invention is to provide a method for the enhanced translocation and/or expression of the products of bacterial fermentation or culture. The nucleic acids described herein can be used to facilitate and increase the synthesis and secretion of products as a result of the enhancement of molecular transport in bacteria when encoded products of such nucleic acids are incorporated into the cellular membranes of bacteria.
Another object of this invention is to provide a membrane transport system which is independent of a naturally-occurring (native) transport system. Thus, prokaryotic or eukaryotic systems can be provided with selected transport mechanisms, especially systems which bypass naturally-occurring transport mechanisms such as the SecA system in E. coli. 
In another embodiment, the DNA of this invention can be used to enhance the growth of nonhuman organisms, and to produce useful quantities of many different substances. These substances include proteins and other molecules which are translocated by cells, as well as substances which are incorporated into cell membranes of all types: i.e., plasma membranes, plastid membranes (including thylakoids), mitochondrial membranes (including cristae), Golgi membranes, endoplasmic reticula membranes, and the like.
Another object of this invention is to provide a vector comprising the DNA of SEQ ID NO:1 or a nucleic acid sequence which hybridizes to SEQ ID NO:1, and a promoter, which vector encodes membrane transport protein Bce44B, or a functional equivalent. Any hybridizing nucleic acid sequence capable of directing protein transport in a manner similar to Bce44B is included.
These vectors can be used in host cells such as prokaryotes and yeasts to enhance transport across cellular membranes. In addition, such vectors can be incorporated into the cells of nonhuman multicellular organisms to enhance translocation of substances across plasma membranes and/or organelle membranes.
Another object of this invention is the enhancement and modification of the import capability of the plastid compartment. Enhancement of protein import may increase the accumulation of all protein products in plastids, particularly in cells of seed and storage tissues. It can also boost the importation of enzymes involved in various biochemical pathways that function within the plastid. General enhancement of protein import in plastids to elevate the amount of biochemical activity in plastids and the storage of proteins and possibly other valuable products can also be achieved using the methods of this invention. Enhancements of this nature can increase product synthesis.
The same benefits can be demonstrated in bacteria using this technology so that products based on bacterial secretion and exportation are more readily accumulated, solubilized and translocated. The chimeric genes and vectors exemplified for both bacteria and plants are available for introduction into selected strains. In fact, the methods of this invention are suitable for use in all prokaryotes and eukaryotes.
This invention also provides a method whereby the activity of the Bce44B protein or its functional equivalent can be or can produce a visual plant transformation marker. Thus, use of markers which are not plant in origin, or herbicide or antibiotic resistance markers which cause concerns about their regulation or widespread use in plants can be avoided.